Human African Trypanosomiasis (HAT, sleeping sickness) is a neglected but fatal vector borne disease caused by Trypanosoma brucei ssp. (T. brucei). Sixty million people are at risk of infection with HAT, and 50,000 new case's and an equal number of deaths are reported annually in subsaharan Africa. The only available anti-trypanosomal drugs on the market are extremely toxic, and result in post-treatment encephalopathy in treated patients. The ideal anti-trypanosomal agents will target vital physiological processes and/or non-variant parasite-derived molecules without adversely affecting the human host. We and others have shown that that cytosolic calcium ion concentration [Ca2+ ]i in blood stages of T. brucei is 4- 10 orders of magnitude below that encountered in host extracellular milieu and that parasites require effective mechanisms to maintain [Ca2+ ]\ homeostasis, survival, and proliferation in their host. In our previous grant, we identified and characterized two key plasma-membrane-like cation pumps (ATPases;TBCA1 and TBCA2) utilized by T. brucei for survival and generated antibodies to immunolocalize them in bloodstage and insect stage parasites. We determined their functional role in insect and bloodstage parasites using transient RNAi inhibition technology and synthetic inhibitor assays. We subsequently constructed recombinant anti-pump vaccines based on a novel bacterial ghost vaccine delivery technology, developed at Morehouse School of Medicine, which partially protected against parasite challenge in mice. Our results revealed that TBCA1 resembled a fungal K+/Na+-ATPase while TBCA2 was a plasma-membrane-like Ca2+ ATPase. RNAi inhibition of these targets resulted in increased parasite mortality. Furthermore, we determined by inhibition studies that Ca2+ homeostasis in T. brucei is not only regulated by the Ca2+ ATPases but also by L-type calcium ion channels. Since targeting the Ca2+ pumps by RNAi inhibition increased parasite mortality and vaccination with TBCA2 significantly reduced parasitemia and survival of infected mice, we propose that targeting the key cation pumps (TBCA1 and TBCA2) as well as L-type Ca2+ channels together with either synthetic inhibitor drugs or vaccines, will be sufficient to inhibit proliferation of T. brucei and provide complete protection against T. brucei infection. In this competitive renewal proposal, we have gone a step further to hypothesize that trypanosomes utilize cation pumps and channels to mediate establishment in mammalian host blood and that simultaneous inhibition by target specific drugs and blocking by vaccination will prevent T. brucei proliferation and development. Two specific aims are proposed: In Specific aim 1. we will functionally characterize and localize the L-type Ca2+ channel in T. brucei and determine its role in Ca2+ homeostasis. In Specific aim 2. we will construct and test various Ca2+ pump/channel gene constructs as antigens in a novel bacterial ghost based vaccine system against infections by T. brucei and determine their levels of protection against T. brucei infection. Our longterm goal is to build on our established proof of principle to develop and deliver a novel class of small molecule drugs and/or immunotherapeutics capable of inhibiting the essential Ca2+ pumps and channels of T. brucei during development. We also plan to generate enough data, through this application, to apply for an RQ-1 type grant to fund future studies on anti-trypanosome drugs.